Five to 8% of the total tRNA populations from Clostridium sticklandii and Methanococcus vannielii contain selenium modified nucleotides. These Se-nucleotides occur in at least 4 to 6 different tRNA species in both organisms. Two of the C. sticklandii selene-tRNAs have been isolated in highly purified forms. The charging activity of several preparations of one of these (tRNA I) was investigated using both homologous and heterologous (E. coli) a.a.-tRNA synthetases. Although Cys, Glu, Met and Trp seemed to be among the most likely candidates, none of these amino acids was detected in ester linkage to tRNA I.